Search

The transcriptional circuits of circadian clocks control physiological and behavioral rhythms. Light may affect such overt rhythms in two ways: (1) by entraining the clock circuits and (2) via clock-independent molecular pathways. In this study we examine the relationship between autonomous transcript oscillations and light-driven transcript responses. Transcript profiles of wild-type and arrhythmic mutant Drosophila were recorded both in the presence of an environmental photocycle and in constant darkness. Systematic autonomous oscillations in the 12- to 48-h period range were detectable only in wild-type flies and occurred preferentially at the circadian period length. However, an extensive program of light-driven expression was confirmed in arrhythmic mutant flies. Many light-responsive transcripts are preferentially expressed in the compound eyes and the phospholipase C component of phototransduction, NORPA (no receptor potential), is required for their light-dependent regulation. Although there is evidence for the existence of multiple molecular clock circuits in cyanobacteria, protists, plants, and fungi, Drosophila appears to possess only one such system. The sustained photic expression responses identified here are partially coupled to the circadian clock and may reflect a mechanism for flies to modulate functions such as visual sensitivity and synaptic transmission in response to seasonal changes in photoperiod., Synopsis Daily changes in sunlight dramatically affect the environment of animals like the fruit fly, a genetic model. To anticipate these changes, the fruit fly possesses a circadian molecular clock that regulates its behavioral activity and other physiology according to the time of day. The clock's mechanism is comprised of genes and their products (proteins and RNAs). One way the clock modulates physiology is by regulating other genes' RNAs. The signature of a circadian RNA is that its levels oscillate once each day. The present study uses microarrays and a novel statistical strategy to resolve how many genes are oscillating in the fly head and verify that there is only a single transcriptional clock. However, this study also finds a large number of genes that are directly regulated by the presence of light. These genes respond to light even in the absence of a functioning clock. Indeed, the light response depends on a signaling component in the retina that has an established role in vision. This represents a new mechanism by which an animal's physiology may be dynamically tuned to daily time.

Accurate predictions of orthology and paralogy relationships are necessary to infer human molecular function from experiments in model organisms. Previous genome-scale approaches to predicting these relationships have been limited by their use of protein similarity and their failure to take into account multiple splicing events and gene prediction errors. We have developed PhyOP, a new phylogenetic orthology prediction pipeline based on synonymous rate estimates, which accurately predicts orthology and paralogy relationships for transcripts, genes, exons, or genomic segments between closely related genomes. We were able to identify orthologue relationships to human genes for 93% of all dog genes from Ensembl. Among 1:1 orthologues, the alignments covered a median of 97.4% of protein sequences, and 92% of orthologues shared essentially identical gene structures. PhyOP accurately recapitulated genomic maps of conserved synteny. Benchmarking against predictions from Ensembl and Inparanoid showed that PhyOP is more accurate, especially in its predictions of paralogy. Nearly half (46%) of PhyOP paralogy predictions are unique. Using PhyOP to investigate orthologues and paralogues in the human and dog genomes, we found that the human assembly contains 3-fold more gene duplications than the dog. Species-specific duplicate genes, or “in-paralogues,” are generally shorter and have fewer exons than 1:1 orthologues, which is consistent with selective constraints and mutation biases based on the sizes of duplicated genes. In-paralogues have experienced elevated amino acid and synonymous nucleotide substitution rates. Duplicates possess similar biological functions for either the dog or human lineages. Having accounted for 2,954 likely pseudogenes and gene fragments, and after separating 346 erroneously merged genes, we estimated that the human genome encodes a minimum of 19,700 protein-coding genes, similar to the gene count of nematode worms. PhyOP is a fast and robust approach to orthology prediction that will be applicable to whole genomes from multiple closely related species. PhyOP will be particularly useful in predicting orthology for mammalian genomes that have been incompletely sequenced, and for large families of rapidly duplicating genes., Synopsis Biologists often exploit the evolutionary relationships between proteins in order to explain how their findings are relevant to the biology of other species, including Homo sapiens. The most natural way to define these relationships is to draw family trees showing, for example, which human protein is the counterpart (“orthologue”) of a protein in dog, and which human proteins have arisen by recent duplication of existing genes (“paralogues”). On a small-scale this is relatively straightforward, but it is difficult to do this automatically on a genome-wide scale. In this paper the authors describe a new approach to drawing a giant family tree of all proteins from humans and dogs. They show how this tree allows them to refine some protein predictions and discard others that are likely to be nonfunctional dead sequences. Family relationships can show how the dog and human genomes have been rearranged since their last common ancestor. In addition, they help to identify the proteins that are specific to either dog or human, and which contribute to these species' biological differences. Giant trees, drawn from this method, will help to associate the differences, duplications, and evolution of proteins in different mammals with their distinctive physiologies and behaviours.

The sequence of the SPF45 protein is significantly conserved, yet functional studies have identified it as a splicing factor in animal cells and as a DNA-repair protein in plants. Using a combined genetic and biochemical approach to investigate this apparent functional discrepancy, we unify and validate both of these studies by demonstrating that the Drosophila melanogaster protein is bifunctional, with independent functions in DNA repair and splicing. We find that SPF45 associates with the U2 snRNP and that mutations that remove the C-terminal end of the protein disrupt this interaction. Although animals carrying this mutation are viable, they are nevertheless compromised in their ability to regulate Sex-lethal splicing, demonstrating that Sex-lethal is an important physiological target of SPF45. Furthermore, these mutant animals exhibit phenotypes diagnostic of difficulties in recovering from exogenously induced DNA damage. The conclusion that SPF45 functions in the DNA-repair pathway is strengthened by finding both genetic and physical interactions between SPF45 and RAD201, a previously uncharacterized member of the RecA/Rad51 protein family. Together with our finding that the fly SPF45 protein increases the survival rate of mutagen-treated bacteria lacking the RecG helicase, these studies provide the tantalizing suggestion that SPF45 has an ancient and evolutionarily conserved role in DNA repair., Synopsis Assigning function to a protein relies on information about similar proteins in different species, and is based on the view that conservation of sequence generally parallels conservation of function. In this article, Chaouki and Salz focus on SPF45, a protein that, at first glance, appears to break this rule. Although the sequence of SPF45 is highly conserved, in animals cells SPF45 functions as a splicing factor, but in plant cells it functions as a DNA repair protein. This functional discrepancy is resolved here through the demonstration that, in D. melanogaster, SPF45 is a bifunctional protein with independent functions in DNA repair and splicing. Support for this conclusion includes the observation that mutant animals lacking SPF45 function display defects in both splicing and DNA repair. In addition, the authors show that SPF45 associates with two distinct groups of proteins; those that participate in RNA splicing and those that participate in DNA repair. The finding that the D. melanogaster protein is bifunctional suggests that the human protein may also have more than one function. This has important clinical implications because elevated SPF45 levels have been correlated with resistance to chemotherapy.

Plants are sessile and therefore have developed mechanisms to adapt to their environment, including the soil mineral nutrient composition. Ionomics is a developing functional genomic strategy designed to rapidly identify the genes and gene networks involved in regulating how plants acquire and accumulate these mineral nutrients from the soil. Here, we report on the coupling of high-throughput elemental profiling of shoot tissue from various Arabidopsis accessions with DNA microarray-based bulk segregant analysis and reverse genetics, for the rapid identification of genes from wild populations of Arabidopsis that are involved in regulating how plants acquire and accumulate Na+ from the soil. Elemental profiling of shoot tissue from 12 different Arabidopsis accessions revealed that two coastal populations of Arabidopsis collected from Tossa del Mar, Spain, and Tsu, Japan (Ts-1 and Tsu-1, respectively), accumulate higher shoot levels of Na+ than do Col-0 and other accessions. We identify AtHKT1, known to encode a Na+ transporter, as being the causal locus driving elevated shoot Na+ in both Ts-1 and Tsu-1. Furthermore, we establish that a deletion in a tandem repeat sequence approximately 5 kb upstream of AtHKT1 is responsible for the reduced root expression of AtHKT1 observed in these accessions. Reciprocal grafting experiments establish that this loss of AtHKT1 expression in roots is responsible for elevated shoot Na+. Interestingly, and in contrast to the hkt1–1 null mutant, under NaCl stress conditions, this novel AtHKT1 allele not only does not confer NaCl sensitivity but also cosegregates with elevated NaCl tolerance. We also present all our elemental profiling data in a new open access ionomics database, the Purdue Ionomics Information Management System (PiiMS; http://www.purdue.edu/dp/ionomics). Using DNA microarray-based genotyping has allowed us to rapidly identify AtHKT1 as the casual locus driving the natural variation in shoot Na+ accumulation we observed in Ts-1 and Tsu-1. Such an approach overcomes the limitations imposed by a lack of established genetic markers in most Arabidopsis accessions and opens up a vast and tractable source of natural variation for the identification of gene function not only in ionomics but also in many other biological processes., Synopsis Unlike most animals, plants are sessile and cannot leave a poor-quality environment after germinating. They therefore need to tolerate the particular conditions they encounter to survive. This makes plants an ideal system for the study of adaptive variation, and this is particularly true of Arabidopsis thaliana (Arabidopsis), which shows substantial natural variation and for which numerous genetic tools exist. Using a combination of analytical chemistry, genetics, and genomics, the authors were able to identify the specific genetic alteration that drive the natural variation in shoot sodium (Na+) accumulation capacity observed in Arabidopsis populations from coastal regions of Spain and Japan (Tossa del Mar and Tsu, respectively). They observed that a deletion in the DNA responsible for regulating the expression of HKT1, a gene known to encode for a Na+ transporter, causes reduced expression of AtHKT1 in roots of both the Spanish and Japanese populations. Such altered expression results in the elevated shoot Na+ observed in these two populations. Interestingly, this novel version of the HKT1 genes is also associated genetically with the enhanced NaCl resistance they observe in the Japanese population.

With mounting availability of genomic and phenotypic databases, data integration and mining become increasingly challenging. While efforts have been put forward to analyze prokaryotic phenotypes, current computational technologies either lack high throughput capacity for genomic scale analysis, or are limited in their capability to integrate and mine data across different scales of biology. Consequently, simultaneous analysis of associations among genomes, phenotypes, and gene functions is prohibited. Here, we developed a high throughput computational approach, and demonstrated for the first time the feasibility of integrating large quantities of prokaryotic phenotypes along with genomic datasets for mining across multiple scales of biology (protein domains, pathways, molecular functions, and cellular processes). Applying this method over 59 fully sequenced prokaryotic species, we identified genetic basis and molecular mechanisms underlying the phenotypes in bacteria. We identified 3,711 significant correlations between 1,499 distinct Pfam and 63 phenotypes, with 2,650 correlations and 1,061 anti-correlations. Manual evaluation of a random sample of these significant correlations showed a minimal precision of 30% (95% confidence interval: 20%–42%; n = 50). We stratified the most significant 478 predictions and subjected 100 to manual evaluation, of which 60 were corroborated in the literature. We furthermore unveiled 10 significant correlations between phenotypes and KEGG pathways, eight of which were corroborated in the evaluation, and 309 significant correlations between phenotypes and 166 GO concepts evaluated using a random sample (minimal precision = 72%; 95% confidence interval: 60%–80%; n = 50). Additionally, we conducted a novel large-scale phenomic visualization analysis to provide insight into the modular nature of common molecular mechanisms spanning multiple biological scales and reused by related phenotypes (metaphenotypes). We propose that this method elucidates which classes of molecular mechanisms are associated with phenotypes or metaphenotypes and holds promise in facilitating a computable systems biology approach to genomic and biomedical research., Synopsis A key challenge of the post-genomic era is to conceive large-scale studies of genomes and observable characteristics of organisms (phenotypes) and to interpret the data thus produced. The goal of this “phenomic” study is to improve our understanding of complex biological systems in terms of their molecular underpinnings. In this paper, Liu and colleagues present comprehensive computational and novel visualization methods for discovering biological knowledge spanning multiple scales of biology. The authors were able to predict and visualize new knowledge between clusters of microbiological phenotypes and their molecular mechanisms. To their knowledge, this is the first time this has been done. More specifically, the method integrates microbiological data with genomic-scale data from protein family databases, gene ontology, and biological pathways. Conducted over 59 fully sequenced bacteria, and including significantly more phenotypes than previous studies of its kind, this study enables a “systems biology” view across different classifications of genes and processes. This represents advancement over previous techniques, which are either limited in biological scale or analytical breadth. Visualization of the networks generated by this technique shows the common biological modules shared by related phenotypes. The results of this experiment demonstrate that the fusion of clinical data with genomic information is able to elucidate, in high throughput, a massive number of biological processes underlying phenotypes.

The LET-60 (Ras)/LIN-45 (Raf)/MPK-1 (MAP kinase) signaling pathway plays a key role in the development of multiple tissues in Caenorhabditis elegans. For the most part, the identities of the downstream genes that act as the ultimate effectors of MPK-1 signaling have remained elusive. A unique allele of mpk-1, ga111, displays a reversible, temperature-sensitive, tissue-specific defect in progression through meiotic prophase I. We performed gene expression profiling on mpk-1(ga111) animals to identify candidate downstream effectors of MPK-1 signaling in the germ line. This analysis delineated a cohort of genes whose expression requires MPK-1 signaling in germ cells in the pachytene stage of meiosis I. RNA in situ hybridization analysis shows that these genes are expressed in the germ line in an MPK-1-dependent manner and have a spatial expression pattern consistent with the location of activated MPK-1. We found that one MPK-1 signaling-responsive gene encoding a C2H2 zinc finger protein plays a role in meiotic chromosome segregation downstream of MPK-1. Additionally, discovery of genes responsive to MPK-1 signaling permitted us to order MPK-1 signaling relative to several events occurring in pachytene, including EFL-1/DPL-1 gene regulation and X chromosome reactivation. This study highlights the utility of applying global gene expression methods to investigate genes downstream of commonly used signaling pathways in vivo., Synopsis In many tissues in developing organisms, signaling pathways interpret extracellular cues that change how genes are expressed inside the nucleus and thus direct the appropriate developmental choice. Identification of the genes that are responsive to signaling pathways is critical for understanding how these pathways can promote the correct cell fate. Additionally, understanding the relationships between different regulatory pathways will also help to decipher the network of gene expression that underlies development. The nematode Caenorhabditis elegans has many signaling pathways that are highly similar to those acting in mammals. In particular, the Ras/Raf/MAP kinase signaling pathway acts in many tissues in C. elegans to direct a diverse set of cell fates. Here, we identify a set of genes whose expression alters in response to Ras/Raf/MAP kinase signaling in the germ line during meiosis. We show that this set of genes is primarily expressed in the germ line and that at least one of these genes is important for proper germ cell fate downstream of Ras/Raf/MAP kinase signaling. We also find that the Ras/Raf/MAP kinase signaling pathway functions independently of a second regulatory pathway, the E2F pathway, that acts at a similar time during germ cell development.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd+) and mutant (Nmd−) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% ± 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd− strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are available., Synopsis Genes determine the structure of proteins through transcription and translation in which an RNA copy of the gene is made (mRNA) and then translated to make the protein. Cellular protein levels reflect the relative rates of mRNA synthesis and degradation, which are subject to multiple layers of controls. Mechanisms also exist to ensure the quality of each mRNA. One quality control mechanism called nonsense-mediated mRNA decay (NMD) triggers the rapid degradation of mRNAs containing coding errors that would otherwise lead to the production of non-functional or potentially deleterious proteins. NMD occurs in yeasts, plants, flies, worms, mice, and humans. In humans, NMD affects the etiology of genetic disorders by affecting the expression of genes that carry disease-causing mutations. Besides quality assurance, NMD plays another role in gene expression by controlling the abundance of hundreds of normal mRNAs that are devoid of coding errors. In this paper, the authors used DNA arrays to monitor the relative decay rates of all mRNAs in budding yeast and found a subset where decay rates were dependent on NMD. Many of the corresponding proteins perform related functional roles affecting both the structure and behavior of chromosomes and the structure and integrity of the cell surface.

Many biological processes are controlled by intricate networks of transcriptional regulators. With the development of microarray technology, transcriptional changes can be examined at the whole-genome level. However, such analysis often lacks information on the hierarchical relationship between components of a given system. Systemic acquired resistance (SAR) is an inducible plant defense response involving a cascade of transcriptional events induced by salicylic acid through the transcription cofactor NPR1. To identify additional regulatory nodes in the SAR network, we performed microarray analysis on Arabidopsis plants expressing the NPR1-GR (glucocorticoid receptor) fusion protein. Since nuclear translocation of NPR1-GR requires dexamethasone, we were able to control NPR1-dependent transcription and identify direct transcriptional targets of NPR1. We show that NPR1 directly upregulates the expression of eight WRKY transcription factor genes. This large family of 74 transcription factors has been implicated in various defense responses, but no specific WRKY factor has been placed in the SAR network. Identification of NPR1-regulated WRKY factors allowed us to perform in-depth genetic analysis on a small number of WRKY factors and test well-defined phenotypes of single and double mutants associated with NPR1. Among these WRKY factors we found both positive and negative regulators of SAR. This genomics-directed approach unambiguously positioned five WRKY factors in the complex transcriptional regulatory network of SAR. Our work not only discovered new transcription regulatory components in the signaling network of SAR but also demonstrated that functional studies of large gene families have to take into consideration sequence similarity as well as the expression patterns of the candidates., Synopsis Many biological processes are controlled by intricate regulatory networks of gene expression. Identifying the regulatory nodes in these networks and understanding the hierarchical relationship between them are vital to our understanding of biological systems. However, this task is frequently hampered by the intrinsic complexity of these processes. Here, the authors used a controlled transcriptional profiling strategy to a plant immune response called systemic acquired resistance to study the transcriptional events one at a time. Systemic acquired resistance is activated through the induction of thousands of genes by the transcriptional regulator protein NPR1. The authors found that downstream of NPR1 are several regulatory nodes comprised of members from a large family of transcriptional factors. Disrupting these regulatory nodes compromised various functions assigned to NPR1, providing the information needed to construct a gene regulation network.

The PMK-1 p38 mitogen-activated protein kinase pathway and the DAF-2–DAF-16 insulin signaling pathway control Caenorhabditis elegans intestinal innate immunity. pmk-1 loss-of-function mutants have enhanced sensitivity to pathogens, while daf-2 loss-of-function mutants have enhanced resistance to pathogens that requires upregulation of the DAF-16 transcription factor. We used genetic analysis to show that the pathogen resistance of daf-2 mutants also requires PMK-1. However, genome-wide microarray analysis indicated that there was essentially no overlap between genes positively regulated by PMK-1 and DAF-16, suggesting that they form parallel pathways to promote immunity. We found that PMK-1 controls expression of candidate secreted antimicrobials, including C-type lectins, ShK toxins, and CUB-like genes. Microarray analysis demonstrated that 25% of PMK-1 positively regulated genes are induced by Pseudomonas aeruginosa infection. Using quantitative PCR, we showed that PMK-1 regulates both basal and infection-induced expression of pathogen response genes, while DAF-16 does not. Finally, we used genetic analysis to show that PMK-1 contributes to the enhanced longevity of daf-2 mutants. We propose that the PMK-1 pathway is a specific, indispensable immunity pathway that mediates expression of secreted immune response genes, while the DAF-2–DAF-16 pathway appears to regulate immunity as part of a more general stress response. The contribution of the PMK-1 pathway to the enhanced lifespan of daf-2 mutants suggests that innate immunity is an important determinant of longevity., Synopsis The innate immune system provides the first line of defense against pathogen infection and relies upon pathways conserved across mammals, insects, and nematodes. Here, the authors have analyzed the transcriptional response of the nematode Caenorhabditis elegans to infection by the human pathogen Pseudomonas aeruginosa. They investigated this transcriptional response in the context of two conserved pathways involved in pathogen defense: the PMK-1 p38 mitogen-activated protein kinase (p38 MAPK) pathway and the DAF-2–DAF-16 insulin-signaling pathway. Specifically, the authors found that the p38 MAPK pathway plays a critical role in the infection-induced expression of secreted immune response genes. These genes include C-type lectins, lysozymes, and antimicrobial peptides that fight off infection in many species. In contrast, they found that the DAF-16 pathway is not required for immune response gene expression and may regulate immunity as part of a general stress response that functions in parallel to p38 MAPK. In addition, the authors observed that p38 MAPK contributes to the enhanced longevity of daf-2 mutants, implicating p38 MAPK signaling in the regulation of longevity, possibly through its role in immunity.

Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the “seed” sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3′ to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide., Synopsis First discovered in nematodes, RNA interference (RNAi) has become an essential tool in the study of mammalian gene function. RNAi directed by small interfering RNAs (siRNAs), 21 nt, double-stranded RNAs target complementary mRNAs for destruction. siRNAs can be introduced into mammalian cells grown in culture, or even administered intravenously to rodents or primates, where they repress production of the targeted gene product. Thus, siRNA-directed RNAi has tremendous potential as a human therapeutic strategy. Dominant genetic disorders, in which a mutant allele of a gene causes disease in the presence of a second, normal copy, might be treated with therapeutic siRNAs, provided that the siRNAs could be designed to destroy the mutant, disease-causing mRNA, while leaving the normal mRNA intact. Here, Schwarz and colleagues describe an experimentally validated strategy for the design of such siRNAs. Their design strategy should facilitate the design of siRNAs targeting dominant genetic disorders such as amyotrophic lateral sclerosis and Huntington disease.

Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5′ splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7–IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5′ pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5′ splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5′ splice site, rather than repressing the 3′ following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5′ pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5′ splice site follows a scanning-type mechanism, precluding competition with other functional 5′ pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5′ pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type–independent 5′−3′-oriented scanning process for accurate recognition of the authentic 5′ splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5′ splice site selection., Synopsis Typically, mammalian genes contain coding sequences (exons) separated by non-coding sequences (introns). Introns are removed during pre-mRNA splicing. The accurate recognition of introns during splicing is essential, as any abnormality in that process will generate abnormal mRNAs that can cause diseases. Understanding the mechanisms of accurate splice site selection is of prime interest to life scientists. Exon–intron borders (splice sites) are defined by short sequences that are poorly conserved. The strength of any splice sequence can be assessed by its degree of homology with a splice site consensus sequence. Within exons and introns, several sequences can match with this consensus as well as or better than the splice sites. Using a system in which a splice site sequence is repeated several times in the intron, the authors showed that linear 5′−3′ search is a leading mechanism underlying splice site selection. This scanning mechanism is cell type–independent, and only the most upstream splice site of all the series is selected, even if splice sites with a better match to the consensus are in the vicinity. These findings reconciliate contradictory observations and establish a hierarchy among the determinants involved in splice site selection.

The packaging of DNA into nucleosomes influences the accessibility of underlying regulatory information. Nucleosome occupancy and positioning are best characterized in the budding yeast Saccharomyces cerevisiae, albeit in asynchronous cell populations or on individual promoters such as PHO5 and GAL1–10. Using FAIRE (formaldehyde-assisted isolation of regulatory elements) and whole-genome microarrays, we examined changes in nucleosome occupancy throughout the mitotic cell cycle in synchronized populations of S. cerevisiae. Perhaps surprisingly, nucleosome occupancy did not exhibit large, global variation between cell cycle phases. However, nucleosome occupancy at the promoters of cell cycle–regulated genes was reduced specifically at the cell cycle phase in which that gene exhibited peak expression, with the notable exception of S-phase genes. We present data that establish FAIRE as a high-throughput method for assaying nucleosome occupancy. For the first time in any system, nucleosome occupancy was mapped genome-wide throughout the cell cycle. Fluctuation of nucleosome occupancy at promoters of most cell cycle–regulated genes provides independent evidence that periodic expression of these genes is controlled mainly at the level of transcription. The promoters of G2/M genes are distinguished from other cell cycle promoters by an unusually low baseline nucleosome occupancy throughout the cell cycle. This observation, coupled with the maintenance throughout the cell cycle of the stereotypic nucleosome occupancy states between coding and non-coding loci, suggests that the largest component of variation in nucleosome occupancy is “hard wired,” perhaps at the level of DNA sequence., Synopsis Every cell contains a complete copy of the genome, which is comprised of DNA. To fit the genome into the cell nucleus, DNA strands are wound around spools made of proteins called histones. By packing DNA, histones also control access to DNA, which influences when genes can be turned on and off. The authors present two main findings. First, they demonstrate a new, simple method to determine how tightly genes are wrapped around histones. Second, they use the new method to determine what happens to DNA packaging at every gene through one cycle of cell growth and division. They find that as genes get turned on throughout the cell cycle, the packaging of DNA that controls those genes becomes loose, and as genes get turned off, it becomes tight again. However, some cell cycle–regulated genes do not fit this pattern, suggesting another means of regulation for these genes. Interestingly, cell cycle–related differences in packaging at single genes are smaller than differences in packaging that are always apparent between different groups of genes. This suggests that most differences in how DNA is packaged may be controlled by the sequence of DNA itself, with smaller contributions from other factors.

The beginning of this millennium has seen dramatic advances in genomic research. Milestones such as the complete sequencing of the human genome and of many other species were achieved and complemented by the systematic discovery of variation at the single nucleotide (SNP) and whole segment (copy number polymorphism) level. Currently most genomics research efforts are concentrated on the production of whole genome functional annotations, as well as on mapping the epigenome by identifying the methylation status of CpGs, mainly in CpG islands, in different tissues. These recent advances have a major impact on the way genetic research is conducted and have accelerated the discovery of genetic factors contributing to disease. Technology was the critical driving force behind genomics projects: both the combination of Sanger sequencing with high-throughput capillary electrophoresis and the rapid advances in microarray technologies were keys to success. MALDI-TOF MS-based genome analysis represents a relative newcomer in this field. Can it establish itself as a long-term contributor to genetics research, or is it only suitable for niche areas and for laboratories with a passion for mass spectrometry? In this review, we will highlight the potential of MALDI-TOF MS-based tools for resequencing and for epigenetics research applications, as well as for classical complex genetic studies, allele quantification, and quantitative gene expression analysis. We will also identify the current limitations of this approach and attempt to place it in the context of other genome analysis technologies. © 2006 Ragoussis et al.

It has been claimed that proteins with more interaction partners (hubs) are both physiologically more important (i.e., less dispensable) and, owing to an assumed high density of binding sites, slow evolving. Not all analyses, however, support these results, probably because of biased and less-than reliable global protein interaction data. Here we provide the first examination of these issues using a comprehensive literature-curated dataset of well-substantiated protein interactions in Saccharomyces cerevisiae. Whereas use of less reliable yeast two-hybrid data alone can reject the possibility that local connectivity correlates with measures of dispensability, in higher quality datasets a relatively robust correlation is observed. In contrast, local connectivity does not correlate with the rate of protein evolution even in reliable datasets. This perhaps surprising lack of correlation with evolutionary rate appears in part to arise from the fact that hub proteins do not have a higher density of residues associated with binding. However, hub proteins do have at least one other set of unusual features, namely rapid turnover and regulation, as manifest in high mRNA decay rates and a large number of phosphorylation sites. This, we suggest, is an adaptation to minimize unwanted activation of pathways that might be mediated by adventitious binding to hubs, were they to actively persist longer than required at any given time point. We conclude that hub proteins are more important for cellular growth rate and under tight regulation but are not slow evolving., Synopsis Why do some proteins evolve so very slowly? Why are only a few proteins uniquely vital to the functioning of an organism? Understanding how proteins interact with other proteins may provide the answers. Some proteins are, it is suggested, like hubs on a wheel with multiple spokes (interacting partners) attached: take away a spoke and the wheel works, take away the hub and the wheel is useless. With so many proteins to bind with, hubs may also be as slow evolving as some interaction sites are constrained in their evolution. Unfortunately, prior analyses have been equivocal, not least because of an uncertainty about which proteins interact with which others. Here the authors employ an extensive literature-curated dataset of reliable protein–protein interactions to address the issue of essentiality, connectivity, and evolutionary rate. This study finds that hubs are more likely to be essential, and if not essential, at least have a larger impact on fitness. However, hub proteins are not slow evolving, in part, because hubs do not have a higher density of binding sites. Hub proteins do, however, appear to be under strong regulation, an adaptation the authors suggest that minimizes the risk of unwanted activation.

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics., Synopsis Amphibian embryos can be used to understand how all vertebrates, including mammals, develop from fertilized single-celled eggs to establish a body plan and form different cell types and functional organs. Genetic methods are used to analyze what goes wrong in embryos lacking working versions of individual genes, and help to understand those genes' specific functions. However, genetic analysis of previously studied amphibians has been difficult because of these species' long generation time and complex genetic structure. The authors have established methods for systematically studying disrupted genes in the frog Xenopus tropicalis, which has a relatively short generation time, simple genetic structure, and an easily studied externally-developing embryo. They describe their methods for creating and characterizing X. tropicalis mutations, using both forward genetics (where a mutation's effects on the embryo are first characterized, then the DNA defect is later identified) and reverse genetics (where animals carrying mutations in a known DNA sequence are first identified, and the effects of that mutation are characterized subsequently). Studies of amphibian development using tissue culture, transplantation, and molecular tools have been fundamental to understanding vertebrate early development. These studies will be greatly enriched by the addition of forward and reverse genetics to complement emerging genomic tools.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

In Caenorhabditis elegans, vulval cell-fate specification involves the activities of multiple signal transduction and regulatory pathways that include a receptor tyrosine kinase/Ras/mitogen-activated protein kinase pathway and synthetic multivulva (SynMuv) pathways. Many genes in the SynMuv pathways encode transcription factors including the homologs of mammalian Rb, E2F, and components of the nucleosome-remodeling deacetylase complex. To further elucidate the functions of the SynMuv genes, we performed a genome-wide RNA interference (RNAi) screen to search for genes that antagonize the SynMuv gene activities. Among those that displayed a varying degree of suppression of the SynMuv phenotype, 32 genes are potentially involved in chromatin remodeling (called SynMuv suppressor genes herein). Genetic mutations of two representative genes (zfp-1 and mes-4) were used to further characterize their positive roles in vulval induction and relationships with Ras function. Our analysis revealed antagonistic roles of the SynMuv suppressor genes and the SynMuv B genes in germline-soma distinction, RNAi, somatic transgene silencing, and tissue specific expression of pgl-1 and the lag-2/Delta genes. The opposite roles of these SynMuv B and SynMuv suppressor genes on transcriptional regulation were confirmed in somatic transgene silencing. We also report the identifications of ten new genes in the RNAi pathway and six new genes in germline silencing. Among the ten new RNAi genes, three encode homologs of proteins involved in both protein degradation and chromatin remodeling. Our findings suggest that multiple chromatin remodeling complexes are involved in regulating the expression of specific genes that play critical roles in developmental decisions., Synopsis In animal cells, DNA and genes are packed into a structure called chromatin. Chromatin-modifying protein complexes play a critical role in the regulation of gene expression. These complexes can alter the chemical and structural properties of the chromosome leading to either the repression or activation of gene expression. How these different complexes coordinate to regulate animal development remains to be explored. Several developmental processes in the nematode Caenorhabditis elegans present excellent model systems to study the functions of chromatin modifications. Using a genome-wide screen, the authors have identified 32 genes that encode potential chromatin-modifying proteins that antagonize the function of another set of transcription regulators including homologs of the mammalian Rb tumor suppressor and components of other chromatin-modifying complexes. The antagonistic roles of these two sets of genes have been observed in a variety of cellular and developmental processes, including organ development and expression of genes in particular tissues. This work indicates that multiple chromatin-modifying complexes are involved in maintaining proper expression of many genes that are critical for precise developmental decisions. Studies on these worm genes should shed light on the roles of the mammalian counterparts in development and related human diseases.

During infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host–pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI) coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies., Synopsis In order to successfully invade and replicate within their hosts, viruses hijack cellular factors. In the case of many RNA viruses, including a Drosophila picorna-like virus Drosophila C virus, they must undergo the essential step of genomic replication on the surface of cytoplasmic membranes. Specifically, for picornaviruses, these vesicles are induced in the infected cell, and the ontogeny and cellular factors required to form this compartment have been unclear. Circumstantial evidence has implicated coat protein complex I (COPI), COPII, and autophagy. Here, Cherry and colleagues present their findings using a genome-wide RNA interference screening approach using a picorna-like virus that COPI and fatty acid biosynthesis are critical host pathways required to generate this intracellular vesicular compartment. Furthermore, they show that loss of COPI, but not COPII, is protective both in adult flies and in human cells infected with the related picornavirus, poliovirus. These novel and exciting findings have broad-scale implications for picornavirus replication and for the potential use of these pathways as novel antiviral targets.

Although large-scale copy-number variation is an important contributor to conspecific genomic diversity, whether these variants frequently contribute to human phenotype differences remains unknown. If they have few functional consequences, then copy-number variants (CNVs) might be expected both to be distributed uniformly throughout the human genome and to encode genes that are characteristic of the genome as a whole. We find that human CNVs are significantly overrepresented close to telomeres and centromeres and in simple tandem repeat sequences. Additionally, human CNVs were observed to be unusually enriched in those protein-coding genes that have experienced significantly elevated synonymous and nonsynonymous nucleotide substitution rates, estimated between single human and mouse orthologues. CNV genes encode disproportionately large numbers of secreted, olfactory, and immunity proteins, although they contain fewer than expected genes associated with Mendelian disease. Despite mouse CNVs also exhibiting a significant elevation in synonymous substitution rates, in most other respects they do not differ significantly from the genomic background. Nevertheless, they encode proteins that are depleted in olfactory function, and they exhibit significantly decreased amino acid sequence divergence. Natural selection appears to have acted discriminately among human CNV genes. The significant overabundance, within human CNVs, of genes associated with olfaction, immunity, protein secretion, and elevated coding sequence divergence, indicates that a subset may have been retained in the human population due to the adaptive benefit of increased gene dosage. By contrast, the functional characteristics of mouse CNVs either suggest that advantageous gene copies have been depleted during recent selective breeding of laboratory mouse strains or suggest that they were preferentially fixed as a consequence of the larger effective population size of wild mice. It thus appears that CNV differences among mouse strains do not provide an appropriate model for large-scale sequence variations in the human population., Synopsis Until recently, it was thought that most inherited human diversity results from genetic variation at single nucleotide sites. However, recent studies discovered many larger-scale differences, involving the duplication or deletion of thousands of bases. Do these large-scale differences contribute greatly to characteristics of human individuals, or are they of little consequence? For clues to solve this mystery the authors looked to the signatures of adaptive evolution written into the DNA. They reasoned that if large-scale DNA differences are beneficial, they should be enriched in genes, particularly those involved in fighting infection and sensing our environment. The authors discovered such enrichments indicating that some large-scale sequence differences have been advantageous during the last approximately 100,000 y of human history. By contrast, modern laboratory mice exhibit few signs of beneficial large-scale DNA differences, perhaps because advantageous sequences have swept rapidly through their ancestral populations. Some large-scale variations in human genomes thus appear to be a legacy of past evolutionary challenges to our species.

Like Muhammad Ali's 1974 “Rumble in the Jungle,” this past fall (September 13–18), the 7th European Meiosis Meeting held in San Lorenzo de El Escorial brought us “Meiosis in Madrid.” This is a sister conference to the Gordon Meiosis series held every other year in the United States, and is an international small-format forum for sharing hot, new results, with an emphasis on unpublished work. At the conference, 21 countries were represented by 167 participants, with a nearly equal split between male and female scientists. The aim of the meeting was to present and promote in-depth discussions about all aspects of meiotic chromosome dynamics, recombination, and segregation. Rather than providing a comprehensive description of the meeting abstracts, this report will briefly describe meeting highlights, guided in large part by feedback from session chairs.

An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets—based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes—provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides a substantially expanded framework for further studies of this model developmental system., Synopsis Animal development requires cells in complex organs to acquire distinct identities. During the development of the body wall musculature of the fruit fly, a pool of apparently identical cells gives rise to two types of muscle precursors, both of which are required for the appearance of functioning muscles. These identities depend on broad programs of gene expression. The authors attempt to dissect the complements of expressed genes that define these two different cell types by integrating modern methods in genetics, genomics, and informatics. By purifying informative cells from normal embryos and mutants that perturb muscle development, assaying their genomewide gene expression programs, and combining experiments statistically, they have identified fivefold more founder-specific genes than were previously suspected to characterize this cell type. The expression patterns of hundreds of genes were examined in whole embryos to test the statistical predictions, permitting the authors to estimate how many more cell type–specific genes remain to be discovered. Finally, dozens of the genes highlighted by these methods were tested for direct involvement in muscle development, and several new players in this process are reported. The integrated strategy used here can be generalized for studying genetic programs in other complex tissues.

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens., Synopsis Ehrlichiosis is an acute disease that triggers flu-like symptoms in both humans and animals. It is caused by a range of bacteria transmitted by ticks or flukes. Because these bacteria are difficult to culture, however, the organisms are poorly understood. The genomes of three emerging human pathogens causing ehrlichiosis were sequenced. A database was designed to allow the comparison of these three genomes to sixteen other bacteria with similar lifestyles. Analysis from this database reveals new species-specific and disease-specific genes indicating niche adaptations, pathogenic traits, and other features. In particular, one of the organisms contains more than 100 copies of a single gene involved in interactions with the host(s). These comparisons also enabled a reconstruction of the metabolic potential of five representative genomes from these bacteria and their close relatives. With this work, scientists can study these emerging pathogens in earnest.

International audience; One of the challenges of genomic research after the completion of the human genome project is to assign a function to all the genes and to understand their interactions and organizations. Among the various techniques, the emergence of chromosome engineering tools with the aim to manipulate large genomic regions in the mouse model offers a powerful way to accelerate the discovery of gene functions and provides more mouse models to study normal and pathological developmental processes associated with aneuploidy. The combination of gene targeting in ES cells, recombinase technology, and other techniques makes it possible to generate new chromosomes carrying specific and defined deletions, duplications, inversions, and translocations that are accelerating functional analysis. This review presents the current status of chromosome engineering techniques and discusses the different applications as well as the implication of these new techniques in future research to better understand the function of chromosomal organization and structures.

Perturbation studies, in which functional performance is measured after deletion, mutation, or lesion of elements of a biological system, have been traditionally employed in many fields in biology. The vast majority of these studies have been qualitative and have employed single perturbations, often resulting in little phenotypic effect. Recently, newly emerging experimental techniques have allowed researchers to carry out concomitant multi-perturbations and to uncover the causal functional contributions of system elements. This study presents a rigorous and quantitative multi-perturbation analysis of gene knockout and neuronal ablation experiments. In both cases, a quantification of the elements' contributions, and new insights and predictions, are provided. Multi-perturbation analysis has a potentially wide range of applications and is gradually becoming an essential tool in biology., Synopsis Which are the important elements of a system? What are their relative contributions to the performance of the various tasks the system is involved in? These simple and basic questions typically arise when analyzing the workings of any system, and of biological systems in particular. In the latter, the elements may be genes, proteins, cells, or tissues, depending on the level and scope of the analysis. To address these questions in a causal manner, perturbations are required, where the elements are perturbed and the resulting performance function is recorded. This approach has been one of the cornerstones of biological research. However, it has usually been confined to the perturbation of a single element at a time, which may lead to misleading results if the elements of the system functionally interact with each other. This paper addresses these questions by providing a quantitative and rigorous method for the analysis of multi-perturbation experiments, where more than one element may be concomitantly perturbed. The workings of the new method are demonstrated in the analysis of genetic multi-knockout experiments of DNA repair in the yeast Saccharomyces cerevisiae and a neural circuit in the nematode Caenorhabditis elegans accounting for chemotaxis. However, the method is general and can be applied to study many other systems in numerous pertinent biological domains.

The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms., Synopsis The pathogenesis of Mycobacterium tuberculosis relies on an ability to survive inside host macrophages. Macrophages kill most other bacteria by engulfment into an intracellular compartment called a phagosome, which quickly matures to an acidic, hydrolytic organelle, resulting in bacterial death. M. tuberculosis and the related vaccine strain M. bovis bacille Calmette-Guérin (BCG) possess the ability to stop phagosome maturation and thus avoid its microbicidal properties. In this study, the researchers screened a library of mutant BCG bacteria to identify the bacterial genes responsible for preventing phagosome acidification. The predicted products of these genes span many different functional groups, but tend to be associated with the outside of the cell or secreted to the extracellular milieu. The researchers also demonstrated that mutant mycobacteria whose phagosomes acidify are unable to replicate in macrophages. This study identifies targets for new vaccines against tuberculosis.

Massive amounts of data are being generated in an effort to represent for the brain the expression of all genes at cellular resolution. Critical to exploiting this effort is the ability to place these data into a common frame of reference. Here we have developed a computational method for annotating gene expression patterns in the context of a digital atlas to facilitate custom user queries and comparisons of this type of data. This procedure has been applied to 200 genes in the postnatal mouse brain. As an illustration of utility, we identify candidate genes that may be related to Parkinson disease by using the expression of a dopamine transporter in the substantia nigra as a search query pattern. In addition, we discover that transcription factor Rorb is down-regulated in the barrelless mutant relative to control mice by quantitative comparison of expression patterns in layer IV somatosensory cortex. The semi-automated annotation method developed here is applicable to a broad spectrum of complex tissues and data modalities., Synopsis The mammalian brain is a complex organ with hundreds of functional parts. Describing when and where genes are expressed in the brain is thus a potentially powerful method for understanding the function of gene products. In recent years, several mammalian genomes including those of human and mouse have been characterized. There are now efforts around the world that aim to determine the expression patterns for all genes in the mouse brain. To search these expression data readily, they must be placed into an atlas. The authors propose a new method for bringing such genetic data into a common spatial framework so that one can perform spatial searches and comparisons of gene expression patterns. To create this atlas, the authors developed a series of maps of the brain using a graphical modeling method called subdivision. These maps were deformed to match the shape of tissue sections, and genetic activity information was associated with the appropriate coordinates on the map. After placing 200 genes into the context of this atlas, the authors illustrate its application in discovering genes potentially involved in diseases and brain development.

An important element of the developing field of proteomics is to understand protein-protein interactions and other functional links amongst genes. Across-species correlation methods for detecting functional links work on the premise that functionally linked proteins will tend to show a common pattern of presence and absence across a range of genomes. We describe a maximum likelihood statistical model for predicting functional gene linkages. The method detects independent instances of the correlated gain or loss of pairs of proteins on phylogenetic trees, reducing the high rates of false positives observed in conventional across-species methods that do not explicitly incorporate a phylogeny. We show, in a dataset of 10,551 protein pairs, that the phylogenetic method improves by up to 35% on across-species analyses at identifying known functionally linked proteins. The method shows that protein pairs with at least two to three correlated events of gain or loss are almost certainly functionally linked. Contingent evolution, in which one gene's presence or absence depends upon the presence of another, can also be detected phylogenetically, and may identify genes whose functional significance depends upon its interaction with other genes. Incorporating phylogenetic information improves the prediction of functional linkages. The improvement derives from having a lower rate of false positives and from detecting trends that across-species analyses miss. Phylogenetic methods can easily be incorporated into the screening of large-scale bioinformatics datasets to identify sets of protein links and to characterise gene networks., Synopsis A typical fully sequenced genome from a bacterial species contains several thousand genes, and those from multicellular animals may contain many thousands of genes. Understanding the function of these genes is one of the key goals of the developing fields of bioinformatics and proteomics, and the results are of interest to life scientists. The authors describe a computational statistical method that can identify pairs of genes whose functions may be linked, in the sense of participating in a common metabolic pathway or from some physical interaction. The method is applied to phylogenetic trees of related organisms and identifies instances in which a pair of genes is either gained or lost together during evolution. They find that genes that have co-evolved like this on two or more occasions during their evolutionary history are almost certainly functionally linked. These methods can be applied in an automated way to large numbers of species for which fully annotated genomes are available to identify candidate sets of functionally linked genes, and to characterize gene networks.

The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups., Synopsis Cells have evolved sophisticated ways to respond to DNA damage. This is critical because unrepaired damage can kill cells or cause them to become cancerous. The response to DNA damage has been studied for more than 50 years, and has been found to be extremely complex. The traditional way of understanding this complexity is to divide the process into its component parts with the goal of eventually reconstituting the entire process. In this study, the authors extend classical approaches using genomics—an approach that involves studying all genes in an organism simultaneously. The authors tested 12 distinct compounds (many used in cancer chemotherapy) that damage DNA and uncovered new genes involved in DNA repair. They then grouped the compounds to define how they attack cells. Using this approach, the study found that many similar DNA-damaging agents act in comparable ways to damage DNA, but surprisingly, similar compounds can also act on cells by very different mechanisms. Specifically grouping the findings together and verifying the significant results lends a high degree of confidence in the data. The development of such a reproducible experimental design is important for inspiring future experiments.

The gene family of subtilisin-like serine proteases (subtilases) in Arabidopsis thaliana comprises 56 members, divided into six distinct subfamilies. Whereas the members of five subfamilies are similar to pyrolysins, two genes share stronger similarity to animal kexins. Mutant screens confirmed 144 T-DNA insertion lines with knockouts for 55 out of the 56 subtilases. Apart from SDD1, none of the confirmed homozygous mutants revealed any obvious visible phenotypic alteration during growth under standard conditions. Apart from this specific case, forward genetics gave us no hints about the function of the individual 54 non-characterized subtilase genes. Therefore, the main objective of our work was to overcome the shortcomings of the forward genetic approach and to infer alternative experimental approaches by using an integrative bioinformatics and biological approach. Computational analyses based on transcriptional co-expression and co-response pattern revealed at least two expression networks, suggesting that functional redundancy may exist among subtilases with limited similarity. Furthermore, two hubs were identified, which may be involved in signalling or may represent higher-order regulatory factors involved in responses to environmental cues. A particular enrichment of co-regulated genes with metabolic functions was observed for four subtilases possibly representing late responsive elements of environmental stress. The kexin homologs show stronger associations with genes of transcriptional regulation context. Based on the analyses presented here and in accordance with previously characterized subtilases, we propose three main functions of subtilases: involvement in (i) control of development, (ii) protein turnover, and (iii) action as downstream components of signalling cascades. Supplemental material is available in the Plant Subtilase Database (PSDB) (http://csbdb.mpimp-golm.mpg.de/psdb.html) , as well as from the CSB.DB (http://csbdb.mpimp-golm.mpg.de)., Synopsis The first complete plant genome sequence was available for Arabidopsis thaliana, a common weed. The number of genes in the Arabidopsis genome is estimated to be around 25,000. The functions of most of these gene are, however, still unknown. Many genes are grouped into gene families due to conserved sequences and predicted protein structures. In this article, the large subtilisin-like serine protease (subtilase) family of Arabidopsis is analysed. Although 56 subtilase genes have been identified in Arabidopsis, the function of only two subtilases is known. Analysis of mutants has revealed no further hints about the function of the other 54 subtilases. Here the authors present a novel approach to infer hypotheses about functions of the subtilase genes using computational analysis. Based on the analyses presented here and in accordance with previously characterized subtilases, they propose three main functions of subtilases: involvement in (i) control of development, (ii) protein degradation, and (iii) signalling. The results presented can be used to direct further analysis to elucidate functions of subtilases in plants.

Mitochondria carry out specialized functions; compartmentalized, yet integrated into the metabolic and signaling processes of the cell. Although many mitochondrial proteins have been identified, understanding their functional interrelationships has been a challenge. Here we construct a comprehensive network of the mitochondrial system. We integrated genome-wide datasets to generate an accurate and inclusive mitochondrial parts list. Together with benchmarked measures of protein interactions, a network of mitochondria was constructed in their cellular context, including extra-mitochondrial proteins. This network also integrates data from different organisms to expand the known mitochondrial biology beyond the information in the existing databases. Our network brings together annotated and predicted functions into a single framework. This enabled, for the entire system, a survey of mutant phenotypes, gene regulation, evolution, and disease susceptibility. Furthermore, we experimentally validated the localization of several candidate proteins and derived novel functional contexts for hundreds of uncharacterized proteins. Our network thus advances the understanding of the mitochondrial system in yeast and identifies properties of genes underlying human mitochondrial disorders., Synopsis Mitochondria are organelles which are best known as the cell's energy powerhouses. They have a special evolutionary origin derived from bacteria engulfed about 2 billion years ago by eukaryotes. Surprisingly, mitochondrial functions have been retained over evolution, so that unicellular yeast and multicellular organisms like humans share many of the same mitochondrial components. Here the authors complemented previous efforts to identify the “parts” of the mitochondrial system, but as for any system, this is not enough to understand how it works. By integrating information on protein localization, function, and interaction, the authors go a step further and propose a map of the mitochondrial organelle and its surroundings. This map suggests the involvement of hundreds of so far uncharacterized proteins in mitochondrial function. By taking advantage of the high conservation of the organelle to humans, the authors investigate properties of human genes involved in mitochondrial diseases. They find that the disease genes have ancient origin and a mild mutant phenotype when their function is abolished in yeast. The approach applied here can be extended to other organelles or organisms and illustrates a growing trend in understanding biological processes in their whole rather than in isolated parts.

Although a large proportion of human transcription occurs outside the boundaries of known genes, the functional significance of this transcription remains unknown. We have compared the expression patterns of known genes as well as intergenic transcripts within the ENCODE regions between humans and chimpanzees in brain, heart, testis, and lymphoblastoid cell lines. We find that intergenic transcripts show patterns of tissue-specific conservation of their expression, which are comparable to exonic transcripts of known genes. This suggests that intergenic transcripts are subject to functional constraints that restrict their rate of evolutionary change as well as putative positive selection to an extent comparable to that of classical protein-coding genes. In brain and testis, we find that part of this intergenic transcription is caused by widespread use of alternative promoters. Further, we find that about half of the expression differences between humans and chimpanzees are due to intergenic transcripts., Synopsis In order to convert the genetic information encoded in an organism's genomic sequence into the functional features, the genomic sequence must be transcribed. According to the current genome annotation, the human genome encodes 20,000–25,000 protein-coding transcripts and a smaller number of non-coding transcripts. There is, however, a growing body of evidence indicating that a much greater proportion of the human genome is transcribed than is accounted for by the existing annotation. Much of this evidence has been found using tiling arrays, microarrays that enable the measurement of transcription regardless of existing annotation. Although some have suggested that these transcripts represent previously unidentified functional RNAs as well as extensions of known genes, the extent of their functionality remains unknown. In this study, Khaitovich et al. assess the functionality of these novel transcripts by testing the extent to which their expression is conserved between humans and chimpanzees in different tissues. The results suggest that, surprisingly, the expression of both known and novel transcripts was affected by the same functional constraints during human and chimpanzee evolution.

Aggressive behavior is important for animal survival and reproduction, and excessive aggression is an enormous social and economic burden for human society. Although the role of biogenic amines in modulating aggressive behavior is well characterized, other genetic mechanisms affecting this complex behavior remain elusive. Here, we developed an assay to rapidly quantify aggressive behavior in Drosophila melanogaster, and generated replicate selection lines with divergent levels of aggression. The realized heritability of aggressive behavior was approximately 0.10, and the phenotypic response to selection specifically affected aggression. We used whole-genome expression analysis to identify 1,539 probe sets with different expression levels between the selection lines when pooled across replicates, at a false discovery rate of 0.001. We quantified the aggressive behavior of 19 mutations in candidate genes that were generated in a common co-isogenic background, and identified 15 novel genes affecting aggressive behavior. Expression profiling of genetically divergent lines is an effective strategy for identifying genes affecting complex traits., Synopsis Aggressive behavior is a complex trait affected by numerous interacting genes whose expression depends on the environment. Aggression can be selectively advantageous in the pursuit of mates, territory, or food; however, excessive aggression may be deleterious. Pathological levels of aggression in humans create an enormous burden to society. Although dysfunction of the biogenic amine systems is often associated with alterations in aggressive behavior, this represents only the “tip of the iceberg” of the complex genetic architecture of aggressive behavior. The fruit fly Drosophila melanogaster is an excellent model genetic system for exploring the genetic basis of aggressive behavior. The authors have developed a rapid assay to quantify Drosophila aggression, and have used it to select genetically divergent replicate lines for increased and decreased behavior from a genetically heterogeneous base population. They used whole-genome expression profiling to identify variation in gene expression among these lines, and identified 1,539 transcripts that differed between the selection lines, illustrating the complex genomic basis of aggressive behavior. The authors evaluated aggressive behavior of flies with mutations in 19 genes that were implicated by the analysis of differential transcript abundance, and identified 15 novel candidate genes affecting this complex trait, eight of which have human orthologs.

In many plant and animal bacterial pathogens, the Type III secretion system (TTSS) that directly translocates effector proteins into the eukaryotic host cells is essential for the development of disease. In all species studied, the transcription of the TTSS and most of its effector substrates is tightly regulated by a succession of consecutively activated regulators. However, the whole genetic programme driven by these regulatory cascades is still unknown, especially in bacterial plant pathogens. Here, we have characterised the programme triggered by HrpG, a host-responsive regulator of the TTSS activation cascade in the plant pathogen Ralstonia solanacearum. We show through genome-wide expression analysis that, in addition to the TTSS, HrpG controls the expression of a previously undescribed TTSS-independent pathway that includes a number of other virulence determinants and genes likely involved in adaptation to life in the host. Functional studies revealed that this second pathway co-ordinates the bacterial production of plant cell wall-degrading enzymes, exopolysaccharide, and the phytohormones ethylene and auxin. We provide experimental evidence that these activities contribute to pathogenicity. We also show that the ethylene produced by R. solanacearum is able to modulate the expression of host genes and can therefore interfere with the signalling of plant defence responses. These results provide a new, integrated view of plant bacterial pathogenicity, where a common regulator activates synchronously upon infection the TTSS, other virulence determinants and a number of adaptive functions, which act co-operatively to cause disease., Synopsis Most pathogenic bacteria have the ability to switch between free-living growth and life within the host tissues. However, the mechanisms that co-ordinate changes in gene expression during the passage between these alternative ecological niches are still largely unknown. A well-studied regulation pathway triggered in response to the host environment is that controlling the transcription of the Type III secretion system (TTSS) genes. The TTSS is a major pathogenicity determinant that delivers bacterial effector proteins directly into the host cell cytosol to promote disease. Here, Valls and colleagues show that the TTSS regulatory pathway is directly connected with other circuits driving the expression of diverse pathogenicity and host-adaptation activities. The authors have identified and characterised the genes co-regulated along with the TTSS via the HrpG regulator. They have found that, in addition to the TTSS, HrpG controls the transcription of a previously unknown TTSS-independent pathway that is essential to pathogenicity and alters the bacterial production of plant cell wall-degrading enzymes, exopolysaccharide, and the phytohormones ethylene and auxin. These findings reveal an important degree of co-ordination between adaptation and virulence functions at the transcriptional level and contribute to a better understanding of the infection process.

The development of DNA microarray technology a decade ago led to the establishment of functional genomics as one of the most active and successful scientific disciplines today. With the ongoing development of immunomic microarray technology-a spatially addressable, large-scale technology for measurement of specific immunological response-the new challenge of functional immunomics is emerging, which bears similarities to but is also significantly different from functional genomics. Immunonic data has been successfully used to identify biological markers involved in autoimmune diseases, allergies, viral infections such as human immunodeficiency virus (HIV), influenza, diabetes, and responses to cancer vaccines. This review intends to provide a coherent vision of this nascent scientific field, and speculate on future research directions. We discuss at some length issues such as epitope prediction, immunomic microarray technology and its applications, and computation and statistical challenges related to functional immunomics. Based on the recent discovery of regulation mechanisms in T cell responses, we envision the use of immunomic microarrays as a tool for advances in systems biology of cellular immune responses, by means of immunomic regulatory network models.

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation., Synopsis The authors have utilized Caenorhabditis elegans, and yeast genetics, combined with mammalian tissue and cell culture experiments to investigate the mechanism of action of a unique set of small molecules. These molecules are active in tissue models of urinary incontinence and allow for increased bladder filling. In the course of studying sensitivity and resistance to these compounds, Fitzgerald et al. uncovered novel alleles of RGS and Gq proteins. Further characterization of one such allele identified that its action conferred a hypo-adaptive phenotype on yeast during pheromone signaling assays. Their data as a whole indicate that these small molecules are able to diminish signaling from G-protein coupled receptors (GPCR) downstream of the receptors themselves. Since GPCR signaling is very important in many diseases in humans, the novel mechanism of these compounds may offer new ways to treat human disease.

Multiple endocrine neoplasia type I (MEN1) is a familial cancer syndrome characterized primarily by tumors of multiple endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein called menin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferase complex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. To elucidate menin's role as a tumor suppressor and gain insights into the endocrine-specific tumor phenotype in MEN1, we mapped the genomic binding sites of menin, MLL1, and Rbbp5, to approximately 20,000 promoters in HeLa S3, HepG2, and pancreatic islet cells using the strategy of chromatin-immunoprecipitation coupled with microarray analysis. We found that menin, MLL1, and Rbbp5 localize to the promoters of thousands of human genes but do not always bind together. These data suggest that menin functions as a general regulator of transcription. We also found that factor occupancy generally correlates with high gene expression but that the loss of menin does not result in significant changes in most transcript levels. One exception is the developmentally programmed transcription factor, HLXB9, which is overexpressed in islets in the absence of menin. Our findings expand the realm of menin-targeted genes several hundred-fold beyond that previously described and provide potential insights to the endocrine tumor bias observed in MEN1 patients., Synopsis In multiple endocrine neoplasia type I, absence of the nuclear factor menin gives rise to endocrine tumors by a mechanism that is poorly understood. Using state-of-the-art genome-wide chromatin-immunoprecipitation coupled with microarray analysis technology, this paper significantly enlarges our understanding of the role of menin by greatly extending the number of gene targets where menin binds. The authors show that while menin frequently colocalizes with a protein complex that modifies chromatin structure, menin can also bind to many other promoters by an alternative mechanism. They also present data that potentially implicate one of the menin target genes, HLXB9, in the endocrine specificity of tumorigenesis in multiple endocrine neoplasia, type 1. Further experiments to confirm the role of HLXB9 in tumorigenesis are necessary and may help explain how the loss of a ubiquitously expressed tumor suppressor gene can give rise to tumors in specific tissues.

CpG island methylation plays an important role in epigenetic gene control during mammalian development and is frequently altered in disease situations such as cancer. The majority of CpG islands is normally unmethylated, but a sizeable fraction is prone to become methylated in various cell types and pathological situations. The goal of this study is to show that a computational epigenetics approach can discriminate between CpG islands that are prone to methylation from those that remain unmethylated. We develop a bioinformatics scoring and prediction method on the basis of a set of 1,184 DNA attributes, which refer to sequence, repeats, predicted structure, CpG islands, genes, predicted binding sites, conservation, and single nucleotide polymorphisms. These attributes are scored on 132 CpG islands across the entire human Chromosome 21, whose methylation status was previously established for normal human lymphocytes. Our results show that three groups of DNA attributes, namely certain sequence patterns, specific DNA repeats, and a particular DNA structure, are each highly correlated with CpG island methylation (correlation coefficients of 0.64, 0.66, and 0.49, respectively). We predicted, and subsequently experimentally examined 12 CpG islands from human Chromosome 21 with unknown methylation patterns and found more than 90% of our predictions to be correct. In addition, we applied our prediction method to analyzing Human Epigenome Project methylation data on human Chromosome 6 and again observed high prediction accuracy. In summary, our results suggest that DNA composition of CpG islands (sequence, repeats, and structure) plays a significant role in predisposing CpG islands for DNA methylation. This finding may have a strong impact on our understanding of changes in CpG island methylation in development and disease., Synopsis DNA methylation is the only epigenetic mechanism in eukaryotes that is known to directly modify the DNA. It plays an important role for gene control during development and cell differentiation, and it is a promising therapeutic target in cancer research. While a genome-wide picture of DNA methylation patterns is currently emerging, we have only fragmentary knowledge about the linkage between DNA methylation and other genomic attributes such as DNA sequence and structure, repetitive elements, or sequence conservation. The authors fill this gap by reporting on a comprehensive bioinformatical analysis of DNA methylation on human Chromosome 21—and in part, extending to other regions of the human genome. They report new associations that will help elucidate the functions of DNA methylation along the human genome. Furthermore, the authors show that their findings can be applied to predicting DNA methylation patterns from genome sequence. Such predictions have the potential of speeding up genome-wide epigenetic profiling: It may be possible to focus experimental resources on a few selected areas while bioinformatics procedures are applied to the bulk of the genome.

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

The subcellular localization of proteins is critical to their biological roles. Moreover, whether a protein is membrane-bound, secreted, or intracellular affects the usefulness of, and the strategies for, using a protein as a diagnostic marker or a target for therapy. We employed a rapid and efficient experimental approach to classify thousands of human gene products as either “membrane-associated/secreted” (MS) or “cytosolic/nuclear” (CN). Using subcellular fractionation methods, we separated mRNAs associated with membranes from those associated with the soluble cytosolic fraction and analyzed these two pools by comparative hybridization to DNA microarrays. Analysis of 11 different human cell lines, representing lymphoid, myeloid, breast, ovarian, hepatic, colon, and prostate tissues, identified more than 5,000 previously uncharacterized MS and more than 6,400 putative CN genes at high confidence levels. The experimentally determined localizations correlated well with in silico predictions of signal peptides and transmembrane domains, but also significantly increased the number of human genes that could be cataloged as encoding either MS or CN proteins. Using gene expression data from a variety of primary human malignancies and normal tissues, we rationally identified hundreds of MS gene products that are significantly overexpressed in tumors compared to normal tissues and thus represent candidates for serum diagnostic tests or monoclonal antibody-based therapies. Finally, we used the catalog of CN gene products to generate sets of candidate markers of organ-specific tissue injury. The large-scale annotation of subcellular localization reported here will serve as a reference database and will aid in the rational design of diagnostic tests and molecular therapies for diverse diseases., Synopsis An important goal of current biological research is annotation of human genes with relevant descriptors and properties. One critical property of interest to biologists and medical researchers is the subcellular localization of gene products, as this affects a protein's biological role and our ability to use it as a therapeutic target. This study used a microarray-based functional genomic method that allows rapid, large-scale identification of subcellular localization, enabling the authors to annotate the localization of thousands of previously uncharacterized human gene products. The authors then provide an example of how these data can be used by applying them to the search for tumor-specific markers. Using data from hundreds of DNA microarray profiles of global gene expression patterns in tumors and normal tissues, they identify candidate genes encoding membrane-associated and secreted proteins that are highly overexpressed in tumors and that might therefore be particularly good targets for diagnostic tests or molecular therapies. Diagnostic tests based on these markers could potentially enable cancers to be detected earlier than is currently possible, and molecular therapies targeting the products of these genes could have high specificity for the corresponding cancers.

During the past ten years, a large body of circumstantial evidence has accumulated, implicating WRKY factors in transcriptional reprogramming during plant immune responses [1–3]. Encoded by complex gene families in higher plants [4], these transcription factors share a DNA-binding domain (WRKY domain) comprising approximately 60 amino acids [5]. Additional conserved features of WRKYs are limited to separate subgroups of this family and include putative leucine zippers, nuclear localization signals, calmodulin binding sites, and several domains of unknown function [5–7]. Multiple studies have demonstrated the ability of WRKYs to bind to promoters of defense-associated genes via specific interactions of their WRKY domains with pathogen response elements termed W boxes (TTGACC/T) [8–11]. Both activating and repressing effects of WRKYs and W boxes on transcription have been observed [12–14]. It has also been shown that stable and transient overexpression of several WRKYs in the model plant Arabidopsis thaliana (Arabidopsis) conveys enhanced resistance to various bacterial or fungal pathogens [15–17].

The Arabidopsis transcription factor HY5 controls light-induced gene expression downstream of photoreceptors and plays an important role in the switch of seedling shoots from dark-adapted to light-adapted development. In addition, HY5 has been implicated in plant hormone signaling, accounting for the accelerated root system growth phenotype of hy5 mutants. Mutants in the close HY5 homolog HYH resemble wild-type, despite the largely similar expression patterns and levels of HY5 and HYH, and the functional equivalence of the respective proteins. Moreover, the relative contribution of HYH to the overall activity of the gene pair is increased by an alternative HYH transcript, which encodes a stabilized protein. Consistent with the enhanced root system growth observed in hy5 loss-of-function mutants, constitutively overexpressed alternative HYH inhibits root system growth. Paradoxically, however, in double mutants carrying hy5 and hyh null alleles, the hy5 root growth phenotype is suppressed rather than enhanced. Even more surprisingly, compared to wild-type, root system growth is diminished in hy5 hyh double mutants. In addition, the double mutants display novel shoot phenotypes that are absent from either single mutant. These include cotyledon fusions and defective vasculature, which are typical for mutants in genes involved in the transcriptional response to the plant hormone auxin. Indeed, many auxin-responsive and auxin signaling genes are misexpressed in hy5 mutants, and at a higher number and magnitude in hy5 hyh mutants. Therefore, auxin-induced transcription is constitutively activated at different levels in the two mutant backgrounds. Our data support the hypothesis that the opposite root system phenotypes of hy5 single and hy5 hyh double mutants represent the morphological response to a quantitative gradient in the same molecular process, that is gradually increased constitutive auxin signaling. The data also suggest that HY5 and HYH are important negative regulators of auxin signaling amplitude in embryogenesis and seedling development., Synopsis Genetic redundancy is the total or partial compensation of inactivation of one gene by another, usually related gene. In Arabidopsis, HY5 and HYH are highly similar, principally exchangeable genes. However, only inactivation of HY5 results in morphological defects, indicating that HY5 plays a more important role in development than HYH. Nevertheless, if inactive versions of such partially redundant genes are combined in a double mutant, the defects observed in the single mutant often worsen. Paradoxically, however, combined inactivation of HY5 and HYH leads to a defect that is opposite to inactivation of HY5 alone: compared to controls, root system growth is decreased in the double mutant, rather than enhanced as in plants only lacking HY5 activity. Through careful analysis of the double mutant defects and scans of genome-wide gene expression levels, the authors determined that the opposite root system growth of hy5 single and hy5 hyh double mutants is a morphological response to a gradually increased quantitative disturbance in the same molecular process, the physiological response to the plant hormone auxin. This example suggests that inactivation of genes that quantitatively affect the balance of a physiological process in the same manner might manifest in very different morphological changes.

Rearrangements of our genome can be responsible for inherited as well as sporadic traits. The analyses of chromosome breakpoints in the proximal short arm of Chromosome 17 (17p) reveal nonallelic homologous recombination (NAHR) as a major mechanism for recurrent rearrangements whereas nonhomologous end-joining (NHEJ) can be responsible for many of the nonrecurrent rearrangements. Genome architectural features consisting of low-copy repeats (LCRs), or segmental duplications, can stimulate and mediate NAHR, and there are hotspots for the crossovers within the LCRs. Rearrangements introduce variation into our genome for selection to act upon and as such serve an evolutionary function analogous to base pair changes. Genomic rearrangements may cause Mendelian diseases, produce complex traits such as behaviors, or represent benign polymorphic changes. The mechanisms by which rearrangements convey phenotypes are diverse and include gene dosage, gene interruption, generation of a fusion gene, position effects, unmasking of recessive coding region mutations (single nucleotide polymorphisms, SNPs, in coding DNA) or other functional SNPs, and perhaps by effects on transvection.

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations., Synopsis Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) is a birth defect with wide geographic distribution, occurring with an average frequency of 1/700 live births. Treatment can be provided, but it will involve medical, surgical, dental, and psychological personnel. Several different genes have been implicated in different cases. Here the researchers report the results of sequencing 20 different genes in 184 CL/P cases selected with an emphasis on more severe cases and cases with a positive family history for CL/P. Genes were selected based on previous work done by others and by the researchers' group. The authors' results suggest that point mutations in these candidate genes are likely to contribute to about 5% of CL/P, and particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and allow investigators to focus functional studies on the rare point mutations that seem to be disease-causing, so that researchers might better understand the mechanisms that play a role in CL/P.

Microbial genes that are “novel” (no detectable homologs in other species) have become of increasing interest as environmental sampling suggests that there are many more such novel genes in yet-to-be-cultured microorganisms. By analyzing known microbial genomic islands and prophages, we developed criteria for systematic identification of putative genomic islands (clusters of genes of probable horizontal origin in a prokaryotic genome) in 63 prokaryotic genomes, and then characterized the distribution of novel genes and other features. All but a few of the genomes examined contained significantly higher proportions of novel genes in their predicted genomic islands compared with the rest of their genome (Paired t test = 4.43E-14 to 1.27E-18, depending on method). Moreover, the reverse observation (i.e., higher proportions of novel genes outside of islands) never reached statistical significance in any organism examined. We show that this higher proportion of novel genes in predicted genomic islands is not due to less accurate gene prediction in genomic island regions, but likely reflects a genuine increase in novel genes in these regions for both bacteria and archaea. This represents the first comprehensive analysis of novel genes in prokaryotic genomic islands and provides clues regarding the origin of novel genes. Our collective results imply that there are different gene pools associated with recently horizontally transmitted genomic regions versus regions that are primarily vertically inherited. Moreover, there are more novel genes within the gene pool associated with genomic islands. Since genomic islands are frequently associated with a particular microbial adaptation, such as antibiotic resistance, pathogen virulence, or metal resistance, this suggests that microbes may have access to a larger “arsenal” of novel genes for adaptation than previously thought., Synopsis More than 250 microbial genomes have been sequenced to date. A significant proportion of the genes in these genomes have no apparent similarity to known genes and their functions are unknown (i.e., they appear to be novel). As the number of sequenced genomes increases, the number of these novel genes continues to increase. In this paper, the authors now show, through an analysis of a diverse range of prokaryotic genomes, that novel genes are more prevalent in regions called genomic islands. Genomic islands are clusters of genes in genomes that show evidence of horizontal origins. This study is notable since genomic islands disproportionately contain many genes of medical, agricultural, and environmental importance (e.g., animal and plant pathogen virulence factors, antibiotic resistance genes, phenolic degradation genes, etc.). The observation that high proportions of novel genes are also localized to genomic islands suggests that microbes may have access to a larger “arsenal” of novel genes for important adaptations than previously thought. These results also imply that there are different gene pools associated with recently horizontally transmitted genomic regions versus regions that are primarily vertically inherited. The authors suggest that further studies involving large-scale environmental genomic sampling are required to help characterize this understudied gene pool.

A region of approximately one megabase of human Chromosome 12 shows extensive linkage disequilibrium in Utah residents with ancestry from northern and western Europe. This strikingly large linkage disequilibrium block was analyzed with statistical and experimental methods to determine whether natural selection could be implicated in shaping the current genome structure. Extended Haplotype Homozygosity and Relative Extended Haplotype Homozygosity analyses on this region mapped a core region of the strongest conserved haplotype to the exon 1 of the Spinocerebellar ataxia type 2 gene (SCA2). Direct DNA sequencing of this region of the SCA2 gene revealed a significant association between a pre-expanded allele [(CAG)8CAA(CAG)4CAA(CAG)8] of CAG repeats within exon 1 and the selected haplotype of the SCA2 gene. A significantly negative Tajima's D value (−2.20, p < 0.01) on this site consistently suggested selection on the CAG repeat. This region was also investigated in the three other populations, none of which showed signs of selection. These results suggest that a recent positive selection of the pre-expansion SCA2 CAG repeat has occurred in Utah residents with European ancestry., Synopsis Natural selection ultimately acts on the genetic variants existing among human populations. Therefore, there are “footprints” that the selective force has left behind in the human genome. In this study, Yu et al. identified an extremely large region on Chromosome 12 that is under positive selection in Utah residents with European ancestry by characterizing the correlation patterns of genomic variants. Further analyses on this interval suggested that selection centered on one of the many forms of Spinocerebellar ataxia type-2 (SCA2) gene. The selected form was next demonstrated to associate with one short version of the disease-causing CAG repeat in the SCA2 gene. These results suggest that the CAG repeat was positively selected. An abnormally long version of CAGs can cause SCA2, a neurodegenerative disease that severely impairs the abilities of body movement. The authors showed how they unraveled natural selection acting on the SCA2 gene. Their findings might lead to the discovery of the biological functions of this gene and its CAG repeat. This kind of study holds potential to facilitate the finding of common disease genes.

Congenital nephrogenic diabetes insipidus (NDI) is a disease characterized by failure of the kidney to concentrate urine in response to vasopressin. Human kindreds with nephrogenic diabetes insipidus have been found to harbor mutations in the vasopressin receptor 2 (Avpr2) gene or the vasopressin-sensitive water channel aquaporin-2 (Aqp2) gene. Development of a treatment is rendered difficult due to the lack of a viable animal model. Through forward genetic screening of ethylnitrosourea-mutagenized mice, we report the identification and characterization of a mouse model of NDI, with an F204V mutation in the Aqp2 gene. Unlike previously attempted murine models of NDI, our mice survive to adulthood and more exactly recapitulate the human disorder. Previous in vitro experiments using renal cell lines suggest recessive Aqp2 mutations result in improper trafficking of the mutant water pore. Using these animals, we have directly proven this hypothesis of improper AQP2 translocation as the molecular defect in nephrogenic diabetes insipidus in the intact organism. Additionally, using a renal cell line we show that the mutated protein, AQP2-F204V, is retained in the endoplasmic reticulum and that this abnormal localization can be rescued by wild-type protein. This novel mouse model allows for further mechanistic studies as well as testing of pharmacological and gene therapies for NDI., Synopsis Nephrogenic diabetes insipidus (NDI) is a disease marked by excessive urination and thirst. Normally, the hypothalamus senses situations where water is limited and signals to the kidney to increase water reabsorption from urine. The signaling molecule secreted by the hypothalamus is arginine vasopressin (AVP), which binds to a specific protein on the surface of kidney cells, AVP receptor (AVPR2). AVP binding to its receptor on kidney cells begins a series of biochemical events that ultimately results in the insertion of a protein, aquaporin 2 (AQP2), into the outer surface of the kidney cell. As its name suggests, AQP2 facilitates the reuptake of water from the urinary space into the cell, thus concentrating the urine and conserving water. Congenital NDI is caused by mutations in either the water channel, AQP2, or in the receptor, AVPR2. While these mutations have been studied extensively in the lab, work in live animals has been very limited. This report describes the first viable mouse model of NDI. Previous models have been attempted by targeted mutation, i.e., genes known to be involved in the disease have been altered in the mouse, a so-called reverse genetic approach. Reverse genetic approaches have so far failed to produce a viable mouse model of NDI. Here the authors take a forward genetic approach in which genes are mutated at random and animals are screened for disease-like properties. As well as proving hypotheses that come from lab studies, this model opens the door to the testing of gene therapy or other therapies for treatment of NDI.

Strand separation is obligatory for several DNA functions, including replication. However, local DNA properties such as A+T content or thermodynamic stability alone do not determine the susceptibility to this transition in vivo. Rather, superhelical stresses provide long-range coupling among the transition behaviors of all base pairs within a topologically constrained domain. We have developed methods to analyze superhelically induced duplex destabilization (SIDD) in genomic DNA that take into account both this long-range stress-induced coupling and sequence-dependent local thermodynamic stability. Here we apply this approach to examine the SIDD properties of 39 experimentally well-characterized autonomously replicating DNA sequences (ARS elements), which function as replication origins in the yeast Saccharomyces cerevisiae. We find that these ARS elements have a strikingly increased susceptibility to SIDD relative to their surrounding sequences. On average, these ARS elements require 4.78 kcal/mol less free energy to separate than do their immediately surrounding sequences, making them more than 2,000 times easier to open. Statistical analysis shows that the probability of this strong an association between SIDD sites and ARS elements arising by chance is approximately 4 × 10−10. This local enhancement of the propensity to separate to single strands under superhelical stress has obvious implications for origin function. SIDD properties also could be used, in conjunction with other known origin attributes, to identify putative replication origins in yeast, and possibly in other metazoan genomes., Synopsis Several DNA functions require the two strands of the DNA duplex to transiently separate. Examples include the initiation of gene expression and of DNA replication. Here the authors examine the strand separation properties of the DNA duplex at autonomously replicating sequences (ARS elements), which are the potential replication origins in yeast. In vivo, susceptibility to strand separation does not depend only on local DNA properties such as adenine plus thymine content or thermodynamic stability. Rather, stresses imposed on the DNA in vivo couple together the strand-opening behaviors of all base pairs that experience them. The authors use computational methods for analyzing stress-driven strand separation to examine the susceptibility to opening of 39 experimentally well-characterized ARS elements. They show that these ARS elements have strikingly increased susceptibilities to stress-induced separation relative to the surrounding sequences. On average, these ARS elements require 4.78 kcal/mol less free energy to separate than do surrounding sequences, making them more than 2,000 times easier to open. This enhanced susceptibility to stress-driven strand separation has obvious implications for the mechanisms that begin the process of replication. This property is also shared by bacterial and viral replication start points, suggesting that it may be a general attribute of replication origins.

Understanding the relationship between protein structure and function is one of the foremost challenges in post-genomic biology. Higher conservation of structure could, in principle, allow researchers to extend current limitations of annotation. However, despite significant research in the area, a precise and quantitative relationship between biochemical function and protein structure has been elusive. Attempts to draw an unambiguous link have often been complicated by pleiotropy, variable transcriptional control, and adaptations to genomic context, all of which adversely affect simple definitions of function. In this paper, I report that integrating genomic information can be used to clarify the link between protein structure and function. First, I present a novel measure of functional proximity between protein structures (F-score). Then, using F-score and other entirely automatic methods measuring structure and phylogenetic similarity, I present a three-dimensional landscape describing their inter-relationship. The result is a “well-shaped” landscape that demonstrates the added value of considering genomic context in inferring function from structural homology. A generalization of methodology presented in this paper can be used to improve the precision of annotation of genes in current and newly sequenced genomes., Synopsis The author provides a novel perspective on a key problem of structural biology: the structure–function relationship in proteins. While relatedness in protein structure correlates with general description of function, attempts to use this relationship predictively are often complicated by its ambiguous nature. A structure encoded by a family of sequences may be implicated in a set of diverse functions across a variety of organisms. The author outlines an innovative approach that underlines the importance of considering genomic context when using structure-comparison methods for functional prediction. First, the author defines two distance measures: in genomic space and in function space. Then, the author describes a landscape of functional distance based on both structural and phylogenetic relatedness. It turns out that this landscape forms a “functional well” where proximity occurs when the structures are similar and occur in the same set of genomes. This result may have implications in future research into functional prediction. With the increasing pace of sequence deposition into databanks, this result suggests a simple way to improve functional prediction via structure homology by complementing existing methods with emerging techniques from comparative genomics.

An important goal of population genetics is to determine the forces that have shaped the pattern of genetic variation in natural populations. We developed a maximum likelihood method that allows us to infer demographic changes and detect recent positive selection (selective sweeps) in populations of varying size from DNA polymorphism data. Applying this approach to single nucleotide polymorphism data at more than 250 noncoding loci on the X chromosome of Drosophila melanogaster from an (ancestral) African population and a (derived) European, we found that the African population expanded about 60,000 y ago and that the European population split off from the African lineage about 15,800 y ago, thereby suffering a severe population size bottleneck. We estimated that about 160 beneficial mutations (with selection coefficients s between 0.05% and 0.5%) were fixed in the euchromatic portion of the X in the African population since population size expansion, and about 60 mutations (with s around 0.5%) in the diverging European lineage., Synopsis The authors provide evidence for the recent action of positive selection in the fruit fly Drosophila melanogaster. They describe a new statistical method to detect footprints of selection in the genome of this species and apply it to a large set of DNA polymorphism data from the X chromosome. They find that numerous selective events occurred in the past 60,000 y, while D. melanogaster expanded its ancestral range in Africa and subsequently colonized temperate zones in the rest of the world after the last ice age. The findings of this paper are important as they indicate where in the genome the genes are located that have been involved in the adaptation of D. melanogaster to environmental changes in the recent past. The approach developed here for the model species D. melanogaster can be readily extended to study adaptation in humans and other species.